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. 2011 Sep;14(3):345-54.
doi: 10.1007/s10456-011-9218-0. Epub 2011 May 29.

TM4SF1: A Tetraspanin-Like Protein Necessary for Nanopodia Formation and Endothelial Cell Migration

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Free PMC article

TM4SF1: A Tetraspanin-Like Protein Necessary for Nanopodia Formation and Endothelial Cell Migration

Andrew Zukauskas et al. Angiogenesis. .
Free PMC article

Abstract

Transmembrane-4-L-six-family-1 (TM4SF1) is a tetraspanin-like membrane protein that is highly and selectively expressed by cultured endothelial cells (EC) and, in vivo, by EC lining angiogenic tumor blood vessels. TM4SF1 is necessary for the formation of unusually long (up to a 50 μm), thin (~100-300 nm wide), F-actin-poor EC cell projections that we term 'nanopodia'. Immunostaining of nanopodia at both the light and electron microsopic levels localized TM4SF1 in a regularly spaced, banded pattern, forming TM4FS1-enriched domains. Live cell imaging of GFP-transduced HUVEC demonstrated that EC project nanopodia as they migrate and interact with neighboring cells. When TM4SF1 mRNA levels in EC were increased from the normal ~90 mRNA copies/cell to ~400 copies/cell through adenoviral transduction, EC projected more and longer nanopodia from the entire cell circumference but were unable to polarize or migrate effectively. When fibroblasts, which normally express TM4SF1 at ~5 copies/cell, were transduced to express TM4SF1 at EC-like levels, they formed typical TM4SF1-banded nanopodia, and broadened, EC-like lamellipodia. Mass-spectrometry demonstrated that TM4SF1 interacted with myosin-10 and β-actin, proteins involved in filopodia formation and cell migration. In summary, TM4SF1, like genuine tetraspanins, serves as a molecular organizer that interacts with membrane and cytoskeleton-associated proteins and uniquely initiates the formation of nanopodia and facilitates cell polarization and migration.

Figures

Fig. 1
Fig. 1
Nanopodia in HUVEC. (A and inset i) HUVEC were plated at 60% confluency for 2h on collagen-I coated glass discs prior to staining with anti-TM4SF1 antibodies and CellMask. CellMask staining of nanopodia was largely continuous and formed occasional lateral membrane bulges (yellow arrow). (B) Transmission immuno-nanogold electron microscopic image of TM4SF1 distribution on apical plasma membrane of a nanopodium of a normal HUVEC. (Lower plasma membrane is not visualized because it remained attached to the glass matrix during processing) Red arrows indicate gold particle clusters (TMED). Scale bar, 100 nm. (C and inset i) GFP-transduced HUVEC were aldehyde-fixed 2h after plating. Intermittently GFP staining nanopodia (white arrows) project from a cell body. Photoshop was used to enhance green color. (D) GFP-transduced HUVEC as in (C) but additionally immunostained with TM4SF1. TM4SF1 commonly co-localized with GFP in nanopodia (yellow arrows). (E, F) Live cell imaging of GFP-transduced HUVEC (time-lapse image frames were selected from Supplemental Video 1). (E) Time-lapse images are shown without (a) or with (b) Photoshop enhancement. Nanopodia (white arrows) extend from short-spike-like projections (red arrows) at the cells’ leading front (0 minute) and trailing rear (127 minutes). (F) Nanopodia that projected from cells #1 and #2 formed transient (white arrows in inset i) or longer lasting contacts that in inset ii (red arrows) persisted for ≥ 12 min. These long lasting contacts may represent nanotubes of the type that have been reported in other types of cultured cells [27]. White scale bars, 10 µm.
Fig. 2
Fig. 2
TM4SF1 overexpressing (OE) HUVEC. HUVEC were transduced with 50 moi adenovirus (empty vector control or full length TM4SF1) for 48h. OE HUVEC expressed TM4SF1 at ~400 mRNA copies/cell. Transduced cells were then subcultured at 60% confluence for 2h (A) or 24h (B) prior to immunostaining with TM4SF1 antibodies, phalloidin, and DAPI. TM4SF1-OE HUVEC projected greatly increased numbers of nanopodia that exhibited nearly continuous TM4SF1 staining (inset ia, white arrows), whereas phalloidin staining was confined to only the most proximal portions of nanopodia (inset ib, pink arrows). (B) By 24h in culture, TM4SF1-OE cells had shed extensive TM4SF1-positive debris that remained attached to substrate (white arrows). (C) Immuno-nanogold electron microscopic staining of TM4SF1 on a nanopodium of TM4SF1-OE HUVEC. Red arrows indicate gold particles. (D) TM4SF1-OE HUVEC performed poorly in an 18h wound healing scratch assay, compared with control, empty vector-transduced HUVEC. Black, white, and yellow scale bars are 0.1 µm, 10 µm and 100 µm, respectively.
Fig. 3
Fig. 3
mRNA expression profiles of L6 family and genuine tetraspanins in HUVEC and HDF. TM4SF1 is strongly expressed in HUVEC but weakly in HDF.
Fig. 4
Fig. 4
HDF form nanopodia and alter their migration pattern following transduction with TM4SF1. HDF were transfected for 48h with empty vector (control) or TM4SF1 adenoviruses and subcultured at 60% confluence for 2h prior to immunostaining (A,B) or live cell imaging (C). (A1) Representative immunofluorescence image of HDF transduced with empty vector at 15 moi demonstrate lack of TM4SF1 staining. (A2) HDF transduced with 15 moi expressed TM4SF1 at ~90 mRNA copies/cell and acquired prominent, broadly rounded lamellipodia from which projected branching (yellow arrows) nanopodia that immunostained with TM4SF1 at a periodicity similar to that of EC nanopodia (inset ia). F-actin was confined proximally (insets ia and ib, pink arrows). (B) HDF expressing ~90 mRNA copies of TM4SF1/cell exhibited an intermittent staining pattern of both TM4SF1 and CD9 with only occasional co-localization (inset i, white arrows). (C) Representative time-lapse images of GFP-transduced HDF that were empty vector control- and TM4SF1-transduced at 15 or 50 moi to express TM4SF1 at ~90 or ~400 mRNA copies/cell. (1) Empty vector (control) transduced HDF exhibited a typical fibroblast migration pattern, projecting relatively narrow, splayed lamellipodia (L, inset i) and changing directions only every 3–5 h. (2) HUVEC transduced with both TM4SF1 (~90 mRNA copies/cell) and GFP projected prominent, larger, and more broadly rounded lamellipodia (L, inset ii) from the leading front and moved continuously in the direction of nanopodia projection (white arrow). (3) TM4SF1 (~400 mRNA copies/cell) and GFP co-transduced HDF projected numerous nanopodia from the entire cell perimeter and movement was impaired. Orange arrows indicate direction of cell migration. White scale bars, 10 µm.
Fig. 5
Fig. 5
TM4SF1 interaction with MYO10 and β-actin in HUVEC. HUVEC were transduced with adenoviruses expressing TM4SF1 (same experimental conditions as in Fig. 2), TM4SF1 knockdown (KD) shRNA, or empty vector (control) [7]. (A) Western blot under reducing (R) or non-reducing (N) conditions stained with TM4SF1 antibody demonstrates three major bands with molecular weights of ~22-, 25-, and 28-kD (black circles). In TM4SF1-KD HUVEC, all three bands were greatly reduced. (B) Cell lysates prepared from 3 day control and TM4SF1-KD HUVEC were pre-absorbed with mouse IgG, immunoprecipitated with anti-TM4SF1 antibodies, and silver stained following SDS-PAGE. Several prominent bands were diminished in TM4SF1-KD lysates. Mass spectrometry (MALDI-TOFF) revealed that band #1 was MYO10 and band #2 was β-actin; sixteen and fourteen peptide bands matched with MYO10 and β–actin, respectively. (C) IP of control HUVEC lysates with TM4SF1 (mouse anti-human), β–actin (rabbit anti-human), or MYO10 (goat anti-human) antibodies, followed by Western blots with anti-TM4SF1 antibodies, consistently demonstrated 28-kD and 25-kD bands. TM4SF1 immunoprecipitates also demonstrated β-actin- and MYO10-reactive bands at their expected molecular weights, 42- and 250-kD, respectively. (D) MYO10 mRNA expression increased 1.8-fold (p=0.004) in TM4SF1-OE HUVEC (~400 TM4SF1 mRNA copies/cell) and decreased by ~30% (p=0.015) in TM4SF1-KD cells. β-actin mRNA levels were not significantly (p=0.17) affected by TM4SF1-OE, but decreased by ~40% (p=0.022) in TM4SF1-KD cells. Student t-test was used to obtain p-values.

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